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In-gel Digestion for Internal Sequences

1. Cut the gel section containing the protein of interest into 1 x 2 mm pieces and place in an eppendorf tube. Repeat for the blank section of gel.

2. Add 250µl 200 mM NH4HCO3 / 50% CH3CN to the gel pieces. (REMOVES CB)

3. Wash for 30 minutes at room temperature on a rocker table.

4. Remove wash and SAVE.

5. Add 100µl NH4HCO3 / 50% CH3CN to the gel pieces.

6. Calculate the volume of 45 mM DTT needed to give a final [DTT] of 1 mM in the sample.

7. Add the above volumes of DTT and then incubate at 37°C for 20 minutes.

8. After incubation, add an equal volume of 100 mM iodoacetic acid (IAA) or 200 mM methyl 4-nitrobenzene sulfonate (MNS) as DTT.

9. For carboxymethyl cysteine (IAA treatment), incubate in the dark for 20 minutes. For S-methyl cysteine (MNS), incubate at 37°C for 40 minutes.

10. After incubation, remove supernatant and SAVE.

11. Repeat steps 2-4.

12. Speedvac gel pieces to complete dryness.

13. Immediately before use, make up a 0.033 mg/ml enzyme stock solution in 200 mM NH4HCO3 by mixing 5.0 µl of 0.1 mg/ml enzyme stock with 10.0 µl 200 mM NH4HCO3.

14. Add a volume of 0.033 mg/ml trypsin or lysyl endopeptidase that equals the initial gel volume (i.e., 15µl per 15 mm3 of gel).

15. Incubate 37°C for 15 minutes. (MAKE SURE GEL IS TOTALLY IMMERSED).

16. If the gel pieces are not totally immersed, add additional enzyme solution until they are just covered.

17. Incubate at 37°C for 24 hours.

18. Extract peptides by adding at least 100µl 0.1% TFA, 60% CH3CN and shaking on a rocker table at room temperature for 60 minutes.

19. Remove supernatant containing peptides and repeat step 18.

20. Speedvac dry the combined washes.

21. Redissolve the dried peptides in 20 µl 0.05% TFA / 25% CH3CN.

22. Sample is ready for reverse phase HPLC separation of peptides on the 173A Microblotter.

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